The proteases may be categorized into families by the different amino acid sequences (generally between 2 and 10 residues) located on either side of the cleavage site of the protease.
The proper functioning of the cell requires careful control of the levels of important structural proteins, enzymes, and regulatory proteins. One of the ways that cells can reduce the steady state level of a particular protein is by proteolytic degradation. Further, one of the ways cells produce functioning proteins is to produce pre or pro-protein precursors that are processed by proteolytic degradation to produce an active moiety. Thus, complex and highly-regulated mechanisms have been evolved to accomplish this degradation.
Proteases regulate many different cell proliferation, differentiation, and signaling processes by regulating protein turnover and processing. Uncontrolled protease activity (either increased or decreased) has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and degenerative disorders.
An additional role of intracellular proteolysis is in the stress-response. Cells that are subject to stress such as starvation, heat-shock, chemical insult or mutation respond by increasing the rates of proteolysis. One function of this enhanced proteolysis is to salvage amino acids from non-essential proteins. These amino acids can then be re-utilized in the synthesis of essential proteins or metabolized directly to provide energy. Another function is in the repair of damage caused by the stress. For example, oxidative stress has been shown to damage a variety of proteins and cause them to be rapidly degraded.
The International Union of Biochemistry and Molecular Biology (IUBMB) has recommended to use the term peptidase for the subset of peptide bond hydrolases (Subclass E.C 3.4.). The widely used term protease is synonymous with peptidase. Peptidases comprise two groups of enzymes: the endopeptidases and the exopeptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively. The term proteinase is also used as a synonym word for endopeptidase and four mechanistic classes of proteinases are recognized by the IUBMB: two of these are described below (also see: Handbook of Proteolytic Enzymes by Barrett, Rawlings, and Woessner AP Press, NY 1998). Also, for a review of the various uses of proteases as drug targets, see: Weber M, Emerging treatments for hypertension: potential role for vasopeptidase inhibition; Am J Hypertens 1999 November; 12(11 Pt 2):139S-147S; Kentsch M, Otter W, Novel neurohormonal modulators in cardiovascular disorders. The therapeutic potential of endopeptidase inhibitors, Drugs R D 1999 April; 1(4):331-8; Scarborough R M, Coagulation factor Xa: the prothrombinase complex as an emerging therapeutic target for small molecule inhibitors, J Enzym Inhib 1998; 14(1):15-25; Skotnicki J S, et al., Design and synthetic considerations of matrix metalloproteinase inhibitors, Ann N Y Acad Sci 1999 Jun. 30; 878:61-72; McKerrow J H, Engel J C, Caffrey C R, Cysteine protease inhibitors as chemotherapy for parasitic infections, Bioorg Med Chem 1999 April; 7(4):639-44; Rice K D, Tanaka R D, Katz B A, Numerof R P, Moore W R, Inhibitors of tryptase for the treatment of mast cell-mediated diseases, Curr Pharm Des 1998 October; 4(5):381-96; Materson B J, Will angiotensin converting enzyme genotype, receptor mutation identification, and other miracles of molecular biology permit reduction of NNT. Am J Hypertens 1998 August; 11(8 Pt 2):138S-142S.
Serine Proteases
The serine proteases (SP) are a large family of proteolytic enzymes that include the hepsin subfamily of proteins, the digestive enzymes trypsin and chymotrypsin, components of the complement cascade and of the blood-clotting cascade, and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. SP are so named because of the presence of a serine residue in the active catalytic site for protein cleavage. SP have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases (cleavage after arginine or lysine), aspases (cleavage after aspartate), chymases (cleavage after phenylalanine or leucine), metases (cleavage after methionine), and serases (cleavage after serine).
A series of six SP have been identified in murine cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells. These SP are involved with CTL and NK cells in the destruction of virally transformed cells and tumor cells and in organ and tissue transplant rejection (Zunino, S. J. et al. (1990) J. Immunol. 144:2001-9; Sayers, T. J. et al. (1994) J. Immunol. 152:2289-97). Human homologs of most of these enzymes have been identified (Trapani, J. A. et al. (1988) Proc. Natl. Acad. Sci. 85:6924-28; Caputo, A. et al. (1990) J. Immunol. 145:737-44). Like all SP, the CTL-SP share three distinguishing features: 1) the presence of a catalytic triad of histidine, serine, and aspartate residues which comprise the active site; 2) the sequence GDSGGP which contains the active site serine; and 3) an N-terminal IIGG sequence which characterizes the mature SP.
The SP are secretory proteins which contain N-terminal signal peptides that serve to export the immature protein across the endoplasmic reticulum and are then cleaved (von Heijne (1986) Nuc. Acid. Res. 14:5683-90). Differences in these signal sequences provide one means of distinguishing individual SP. Some SP, particularly the digestive enzymes, exist as inactive precursors or preproenzymes, and contain a leader or activation peptide sequence 3′ of the signal peptide. This activation peptide may be 2-12 amino acids in length, and it extends from the cleavage site of the signal peptide to the N-terminal IIGG sequence of the active, mature protein. Cleavage of this sequence activates the enzyme. This sequence varies in different SP according to the biochemical pathway and/or its substrate (Zunino et al, supra; Sayers et al, supra). Other features that distinguish various SP are the presence or absence of N-linked glycosylation sites that provide membrane anchors, the number and distribution of cysteine residues that determine the secondary structure of the SP, and the sequence of a substrate binding sites such as S′. The S′ substrate binding region is defined by residues extending from approximately +17 to +29 relative to the N-terminal I (+1). Differences in this region of the molecule are believed to determine SP substrate specificities (Zunino et al, supra).
The human hepsin cDNA was initially isolated from a liver cDNA library screened with a mixture of oligonucleotides based on a consensus sequence of serine proteases, Leytus et al., Biochemistry 27:1067-1074 (1988). Biochemical studies indicate that hepsin is a type II transmembrane serine protease expressed mainly on the surface of hepatocytes, Tsugi et al., J. Biol. Chem. 266:16948-16953 (1991). Lower levels of hepsin mRNA are detected in other tissues including lung, kidney, pancreas, stomach, thyroid and prostate. In addition, hepsin mRNA is present in several human tumor cell lines, such as hepatoma cells HepG2 and PLC/PRF/5, mammary cancer cells MCF784 and T470, and epitheloid carcinoma cells HeLa S3, Tsuji et al., J. Biol. Chem. 266:16948-16953 (1991) and Torres-Rosado et al., Proc. Natl. Acad. Sci. USA 90:7181-7185 (1993).
Hepsin has a number of reported activities. In an in vitro study, recombinant human hepsin expressed on the cell surface activated blood coagulation factor VII but not factors IX, X, prothrombin or protein C, all of which share significant structural and sequence similarities with factor VII. The activation of factor VII by hepsin was shown to be sufficient to initiate the coagulation pathway leading to thrombin formation, Kazama et al., J. Biol. Chem. 270:66-72 (1995). Elevated plasma factor VIIa activity has been known to be a significant risk factor for ischemic heart disease and cardiovascular death, Hultin, Prog. Hemostasis Thrombosis 10:215-241, (1991) and Mann, Arteriosclerosis 9:783-784 (1989). Factor VIIa/tissue factor complex also contributes to tumor-related hypercoagulability and intravascular thrombosis, Edwards et al., Thromb. Haemostasis 69:205-213 (1993).
In addition to blood coagulation, hepsin was reported to be critical for cell growth. In a cell culture system, addition of anti-hepsin antibodies or hepsin-specific antisense oligonucleotides to the culture medium significantly inhibited growth of hepatoma cells, Torres-Rosado et al., Proc. Natl. Acad. Sci. USA, 90:7181-7185 (1993). This observation is quite interesting in light of the expression of hepsin mRNA in a number of tumor cells.
The growth factor-like activities of serine proteases have been known for many years. For example, thrombin is a potent mitogen for vascular fibroblasts and smooth muscle cells, Fenton J, Ann. N.Y. Acad. Sci., 485:5-15 (1986). Furthermore, serine proteases also participate in processing of growth factors, Massague J, J. Biol. Chem., 265:21393-21396 (1990). The hepsin-dependent tumor cell growth indicates a mechanism in which hepsin functions either directly as a growth factor or indirectly as an enzyme that processes certain growth factors essential for cell growth.
For a review of hepsin, see Tsuji et al., J. Biol. Chem. 266:16948-16953, 1991; Tanimoto et al., Cancer Res. 57: 2884-2887, 1997; Zacharski, Thromb. Haemost. 79:876-877, 1998; Torres-Rosado et al., Proc. Natl. Acad. Sci. 90:7181-7185, 1993; Kazama et al., J. Biol. Chem. 270: 66-72, 1995; Luo et al., Cancer Res. 61: 4683-4688, 2001; Magee et al., Cancer Res. 61:5692-5696, 2001; Welsh, Cancer Res. 61: 5974-5978, 2001; Dhanasekaran et al., Nature 412: 822-826, 2001; Stamey et al., J. Urol. 166:2171-2177, 2001; Vu et al., J. Biol. Chem. 272:31315-31320, 1997.
Protease proteins, particularly hepsin, a member of serine protease subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of protease proteins. The present invention advances the state of the art by providing a previously unidentified human hepsin protein.